1. Field of the Invention
The invention relates to fractionation apparatus and methods, and finds especially advantageous application to the separation of biological macromolecules.
2. Description of the Related Art
The separation and purification of biological macromolecules is an important aspect of biochemical research and pharmaceutical development. Accordingly, a wide variety of procedures have been developed to isolate desired molecular species from a crude sample of biological material such as a cell lysate. These methods include electrophoresis, elution through a chromatography column, field-flow fractionation, and sequential precipitation techniques, to name a few. In most instances, performing a series of several different separation steps is required in the process of isolating a desired molecule from an initial sample of raw biological material.
Among the various separation protocols mentioned above, the sequential precipitation techniques are both commonly performed and labor intensive. One specific precipitation technique, ammonium sulfate precipitation, has been estimated to be part of approximately 80% of the currently developed protein purification protocols. In this technique, purification is performed by sequentially precipitating sample fractions in solutions having either a progressively higher or a progressively lower concentration of ammonium sulfate. This process requires a large amount of tedious manual preparation of ammonium sulfate solutions as well as manual fraction collection and re-precipitation.
Thus, in contrast with many other molecular separation techniques which may be relatively highly automated, precipitation based separation methods remain labor intensive and highly time consuming.
The invention provides a significant increase in the speed and efficiency of precipitation based fractionation apparatus and methods. In one embodiment, the invention comprises a channel having therein a solution of a precipitation reagent, wherein the concentration of the precipitation reagent is different at a first position in the channel than at a second position in the channel. The concentration at any given point in the channel may be reduced or increased as a function of time.
The apparatus may take a variety of forms. In one embodiment, the apparatus comprises a first channel containing a precipitation reagent, a second channel containing a sample for fractionation, and a semi-permeable membrane separating the first channel from the second channel.
Methods of separating target entities are also provided. In one embodiment, such a method comprises varying a concentration of a precipitation reagent from a first end of a channel to a second end of a channel, introducing a sample into the first end of the channel, and eluting the sample from the second end of the channel.